Noonan syndrome (NS) is a common (1 in 1500 to 2500 live births) autosomal-dominant disorder caused by mutations in PTPN11 (50%), SOS1 (20%), or KRAS (<5%) and is characterized by dysmorphic facial features, congenital heart malformations, skeletal anomalies, and a variety of hematologic abnormalities including a predisposition to juvenile myelomonocytic leukemia (JMML). Although the anomalies observed in NS are diverse, several of the complications can be attributed to the increased function or number of macrophages or monocyte-derived cells. The most extreme and lethal example of macrophage overproduction in NS patients is the childhood leukemia, JMML. JMML is characterized clinically by overproduction of myelomonocytic cells and by the in vitro phenotype of hematopoietic progenitor hypersensitivity to granulocyte-macrophage colony- stimulating factor (GM-CSF). In addition to GM-CSF-stimulated hyperproliferation and elevated levels of phospho-Erk, we found that activating Shp2 mutations (Shp2E76K and Shp2D61Y) promote a shift toward monocytic differentiation at the expense of other myeloid lineages, consistent with the disease phenotype observed in JMML patients. This skewed differentiation toward the monocytic lineage is associated with increased c-Jun and decreased GATA2 expression. Based on these findings, we hypothesize that activating Shp2-induced Ras hyperactivation alters the transcriptional profile leading to enhanced monocytic differentiation at the expense of alternative myeloid lineages. Mechanistically, we propose that Ras hyperactivation produces constitutive c-Jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression. The objectives of this application are to 1) delineate if JMML associated Shp2 gain-of-function mutations induce c-Jun expression and promote aberrant monocytic differentiation in a JNK-dependent or a JNK-independent manner;2) investigate the potential of trichostatin A, a histone deacetylase inhibitor, to reduce c-Jun and PU.1 and to re-activate GATA2 and C/EBP? expression in mutant Shp2-expressing hematopoietic progenitors;and 3) examine hematopoietic- specific transcription factor levels in human samples of JMML compared to normal controls. We anticipate that results of these studies will lead to novel therapeutic strategies in JMML, a lethal childhood leukemia and complication of Noonan syndrome. PUBLIC HEALTH RELEVANCE: This proposal, "Aberrant Monocytic Differentiation Induced by Gain-of-Function Shp2 Mutants" focuses on a particularly lethal form of childhood myeloid leukemia called juvenile myelomonocytic leukemia (JMML). JMML is a lethal leukemia of children less than 5 years of age characterized by massive overproduction of monocytic cells. Unfortunately, JMML is resistant to chemotherapy and most afflicted children succumb to disease due to organ infiltration with malignant monocytes and macrophages, ending terminally in bleeding and infection. Experiments outlined in this proposal are directed at identifying the molecular mechanisms that enhance monocyte overproduction in order to delineate novel targets for therapeutic intervention in JMML.